JP4096037B2 - Prediction method of drug metabolic activity by mutation analysis of glucuronyltransferase gene - Google Patents

Description

反応物はエタノール沈殿法により精製した。
【００２６】
【実施例３】
（ハイブリダイゼーションおよび検出）
実施例２で調製した各試料を１２μＬのハイブリダイゼーション緩衝液〔４×ＳＳＣ（０．１５ｍｏｌ／ＬのＮａＣｌと１５ｍｍｏｌ／Ｌのクエン酸ナトリウムを基本溶液として４×はその４倍濃度を表す）、０．２％ＳＤＳ、５０％ホルムアミド〕に溶解し、その１０μＬを実施例１で作成した核酸アレイに４２℃で２時間ハイブリダイズさせた。ハイブダイズ後、緩衝液（２×ＳＳＣ、０．２％ＳＤＳ）で３７℃、５分間洗浄後さらに、緩衝液（０．２×ＳＳＣ）で室温、５分間洗浄後、乾燥し、Ａｆｆｉｍｅｔｒｉｘ ４２８ ＡｒｒａｙＳｃａｎｎｅｒを用いて蛍光シグナルを検出した。その結果を図２〜８に示した。
【００２７】
試料１では、Ｇ７１Ｒの変異検出チップのＧ列のみに、Ｐ２２９Ｑ変異検出チップのＣ列のみに、さらにＹ４８６Ｄの変異検出チップのＴ列のみにそれぞれハイブリダイズが観察された。これは、何れも正常な配列を有していることを示していた（図２）。
【００２８】
試料２および３では、Ｐ２２９Ｑの変異検出チップのＣ列およびＹ４８６Ｄの変異検出チップのＴ列にハイブリダイズが認められ、それぞれ正常な配列が認められたが、Ｇ７１Ｒの変異検出チップのＡ列およびＧ列の両方にハイブリダイズが観察された。これにより、Ｇ列にハイブリダイズする正常な配列と、Ａ列にハイブリダイズする変異配列を共に有するヘテロ接合性の変異が確認された（図３、図４）。
【００２９】
試料４においては、Ｐ２２９ＱおよびＹ４８６Ｄの変異検出チップでは何れも正常なハイブリダイズパターンを示したが、Ｇ７１Ｒの変異検出チップのＡ列のみにハイブリダイズが観察された。これにより、ホモ接合性の変異が確認された（図５）。
【００３０】
試料５では、Ｇ７１ＲおよびＹ４８６Ｄの変異検出チップのハイブリダイズパターンは何れも正常であったが、Ｐ２２９Ｑにおいては正常なハイブリダイズであるＣ列とさらにＡ列にもハイブリダイズが観察された。これにより、ヘテロ接合性の変異が確認された（図６）。
【００３１】
試料６ではＧ７１ＲおよびＰ２２９Ｑの変異検出チップに対しては何れも正常なハイブリダイズパターンを示したが、Ｙ４８６Ｄの変異検出チップに対しては、正常なハイブリダイズであるＴ列と変異を示すＧ列の両方にハイブリダイズが観察された。これにより、ヘテロ接合性の変異が確認された（図７）。
【００３２】
試料７では試料６と異なり、Ｙ４８６Ｄ変異検出チップにおいてＧ列のみにハイブリダイズが観察された。これにより、ホモ接合性の変異が確認された（図８）。
【００３３】
以上７種の試料と同じ遺伝子配列を持つＵＧＴ分子を遺伝子組換えにより調製し、ビリルビンを基質として測定したときのＵＧＴ活性と上記の結果を比較検討した結果、表１のようになり、ＵＧＴ分子をコードするエキソン５領域の変異、とりわけＹ４８６Ｄの変異を検出することにより、ＵＧＴ分子の酵素活性を予測することが可能なことが確認された。これより薬剤代謝の異常を検査することが可能であることが示された。さらに、エキソン５領域以外のエキソン１〜４の領域の変異を合わせて検出することにより、より効果的に薬剤代謝の異常を検査できることが示された。
【００３４】
（表１）ＤＮＡチップでの解析結果と対応する遺伝子組換えＵＧＴ１Ａ１分子のＵＧＴ活性
【００３５】
【表１】

【００３６】
【実施例４】
実施例１と同様に操作してエキソン５領域の変異を検出したＵＧＴ１Ａ６分子について、エキソン５領域の変異の解析結果と、対応するその遺伝子組換えＵＧＴ１Ａ６分子のＵＧＴ活性を２−アミノ−５−ニトロ−４−トリフルオロメチルフェノールを基質として測定した。その結果を表２に示した。
【００３７】
（表２）ＤＮＡチップでの解析結果と対応する遺伝子組換えＵＧＴ１Ａ６分子のＵＧＴ活性
【００３８】
【表２】

【００３９】
この結果、ＵＧＴ１Ａ１のみならず、他のアイソフォームの変異も本発明の方法で検出可能であることが判り、本発明の効果が確かめられた。
【００４０】
【発明の効果】
以上説明したように、本発明のＵＧＴ１をコードする共通エキソン領域の変異を検出方法により、多数の存在するアイソフォームを個別に検査することなく、効率的にＵＧＴ１遺伝子の変異を検出して、数多い薬剤に対してその代謝の判定、予測および検査を効率的に行うことができる。
【００４１】
【図面の簡単な説明】
【図１】ＤＮＡチップの核酸プローブの配置を示した図である。
【図２】試料１（正常検体）の検査結果を表した図である。
【図３】試料２の検査結果を表した図である。
【図４】試料３の検査結果を表した図である。
【図５】試料４の検査結果を表した図である。
【図６】試料５の検査結果を表した図である。
【図７】試料６の検査結果を表した図である。
【図８】試料７の検査結果を表した図である。
【配列表】

[0001]
BACKGROUND OF THE INVENTION
The present invention is used in the field of clinical testing, and more particularly to a method for genetic testing of an enzyme involved in glucuronidation, and a probe and kit therefor.
[0002]
[Prior art]
Uridine diphosphate glucuronosyltransferase (UDP) is an enzyme that catalyzes the glucuronidation of various drugs. UGTs are classified into two families, UGT1 and UGT2, based on the homology of their amino acid sequences.
It is known that UGT1 has at least nine isoforms, UGT1A1 and UGT1A3 to UGT1A10. For example, UGT1A1 conjugates bilirubin, amine, phenol and the like, and UGT1A6 conjugates phenol having a planar molecular structure. The human UGTl gene (UGT1) is present on chromosome 2q37 and consists of exon 1 having substrate specificity for each isoform (1A1 to 1A10) and exons 2 to 5 common to each isoform. A promoter region containing TATAbox is present upstream. Each isoform has a unique amino terminal region encoded by at least one of the first exon group and a common carboxy terminal region encoded by four exons. Therefore,
On the other hand, UGT2 is divided into UGT2A conjugated with odorous substances, UGT2 conjugated with bile acids and steroids.
[0003]
Other than high serum bilirubin, general liver function tests are normal, and no obvious cause of jaundice (such as hemolysis) is called constitutional jaundice, and indirect (unconjugated) bilirubin is elevated. -Najjar syndrome type I, type II, and Gilbert syndrome are divided roughly into Dubin-Johnson syndrome and Rotor syndrome in which direct (conjugated) bilirubin is elevated. In Crigler-Najjar syndrome type I, type II and Gilbert syndrome, mutations in exon 5 of the UGT1A1 gene have been reported. Specifically, a mutation (Y486D) in which the tyrosine at amino acid sequence number 486 is replaced with aspartic acid reduces the enzyme activity to 13 times that of a normal one.
[0004]
On the other hand, cytochrome P450 is well known as a substance involved in drug metabolism in the body. It is also well known that due to the difference in the polymorphism of this enzyme, a certain drug is not metabolized and a drug metabolism disorder is caused. Drug metabolism abnormalities are not only caused by cytochrome P450 polymorphism, but also as described above, since the drug is metabolized by glucuronidation, the UGT polymorphism is known to be involved in drug metabolism. However, in the relationship between various drug metabolisms and UGT polymorphisms, effective gene mutations that can reflect the metabolism for many drugs are not clear. In particular, the relationship between the mutation of exon 5 region of UGT1 gene and drug metabolism is not clear.
[0005]
[Problems to be solved by the invention]
An object of the present invention is to provide a method for determining, predicting or testing drug metabolism by efficiently detecting a mutation in a gene encoding UGT.
[0006]
[Means for solving the problems]
As a result of intensive studies, the present inventors have found that a gene encoding UGT is composed of five exons, and the exons 2 to 5 region are common to each isoform of UGT1, and therefore, mutations in these regions In particular, focusing on the fact that the mutation of UGT1 can be detected effectively without examining each isoform of UGT1, especially by detecting the mutation in exon 5 region of UGT1 molecule, the prediction of drug metabolism Has been found to be effective, and the present invention has been completed.
[0007]
That is, the present invention
1. A test method for drug metabolizing ability of UGT, comprising a step of detecting a mutation in exon 5 region of a gene encoding UDP-glucuronosyltransferase (UGT);
2. The test method according to item 1, comprising a step of detecting a mutation in the promoter region,
3. A sample containing a gene encoding UGT1 is tested for each isoform of UGT1A1, UGT1A3, UGT1A4, UGT1A5, UGT1A6, UGT1A7, UGT1A8, UGT1A9 and UGT1A10. 3. The inspection method according to item 1 or 2, comprising a step of detecting a mutation in exon 5 region,
4). The test method according to item 3 above, which comprises a step of detecting a mutation in the exon 5 region of each isoform of the UGT1A molecule corresponding to the 1456th base of the UGT gene sequence encoding the 486th amino acid of the UGT1A1 molecule,
5. 5. The examination according to any one of the preceding items 1 to 4, which comprises a step of detecting a mutation in at least one region of exons 1, 2, 3 and 4 of a gene sequence encoding a UGT molecule together with the mutation detection step. Method,
6). A step of detecting a mutation in at least one gene sequence among the 226th mutation in the UGT gene sequence encoding the 71st amino acid of the UGT1A1 molecule and the 486th mutation in the gene sequence encoding the 229th amino acid. The inspection method according to item 5 above, comprising
7). A UGT gene having a mutation consisting of the base substitution according to the above item 3 or 4, or a gene fragment containing the mutation,
8). In order to use a DNA fragment having a functional effective length as a test DNA used in the method for detecting base substitution according to any one of items 1 to 6 or the base substitution according to items 1 to 6 in the detection method A DNA fragment having a functional effective length as a probe of
9. 9. The DNA fragment according to item 7 or 8 above, which is an oligonucleotide probe specific for UGT having the base sequence represented by any one of SEQ ID NOs: 1 to 3.
10. 7. The inspection method according to item 5 or 6, wherein the probe having the base sequence represented by SEQ ID NO: 1 and the probe having the base sequence represented by SEQ ID NO: 2 and / or 3 are used in combination.
11. The detection apparatus which installed the oligonucleotide probe used for the method of Claim 10 or the oligonucleotide probe of any one of Claims 7-9 in the same apparatus.
12 12. The detection according to item 11 above, wherein the end of the base sequence of the oligonucleotide probe according to any one of items 7 to 9 is a nucleic acid chip or a nucleic acid array immobilized by binding to an insoluble support through a functional group. Device 13. 13. A method for determining, predicting or examining drug metabolism using the apparatus according to 11 or 12 above.
14 The test kit is used for the method according to any one of items 1 to 6, 10, and 13, or the nucleic acid fragment according to items 7 to 9 or the test kit incorporating the apparatus according to item 11 or 12.
[0008]
[Embodiments of the present invention]
In order to describe the present invention in detail, an embodiment will be illustrated and described.
2-Amino-5-nitro-4-trifluoromethylphenol glucuronic acid conjugate is the main metabolite of flutamide, a non-steroidal anti-androgenic agent used in the treatment of prostate cancer with strong hepatotoxicity.
2-Amino-5-nitro-4-trifluoromethylphenol is glucuronidated by UGT. Enzymatic reaction when glucuronidation was carried out by the enzyme of each isoform of UGT1A1 and UGT1A6 was compared between a normal molecule and a molecule having a Y486D mutation. As a result, the UGT1A1 mutant showed a maximum rate of about 12% compared to the maximum rate of the natural type, and the Km value was about half that of 2-amino-5-nitro-4-trifluoromethylphenol. , UDP-glucuronic acid was equivalent to the natural type. On the other hand, for UGT1A6, the mutant type showed only 1% or less of the activity of the natural type, and the maximum reaction rate and Km could not be measured.
As described above, the UGT1 gene has a common exon 2-5 region for each isoform and a different exon 1 region for each isoform. Therefore, when a mutation occurs in the common exon region, the enzyme activity of UGT1 is increased in all isoforms. It can be said to decline.
[0009]
The present invention provides a method for efficiently examining gene mutations for all isoforms that may exist in UGT1, by examining mutations in the common exon region. Specifically, for example, by examining a mutation (Y486D) in the exon 5 region of UGT1A1 and UGT1A6, drug metabolism by these enzymes can be predicted.
Furthermore, the present invention can detect mutations unique to each isoform by adding mutation detection in the exon 1 region of UGT1 and combine them to reduce the frequency of undetected UGT gene mutations overall. A UGT gene mutation test method is provided. This can provide a method for efficiently predicting and examining drug metabolism.
[0010]
The present invention discloses a method for testing a mutation in the exon 5 region of UGT1A1, specifically the Y486D mutation, and this test method can also detect other isoform mutations occurring simultaneously. For example, UGT1A3, UGT1A4 and UGT1A5 mutation Y487D, UGT1A6 mutation Y485D, and UGT1A7, UGT1A8, UGT1A9 and UGT1A10 mutation Y483D are tested using the same probe and / or the same apparatus. be able to. Therefore, by using the method of the present invention, all the exons of these isoforms can be obtained by using one nucleic acid probe without individually examining the mutation of each isoform of UGT1A that plays an important role in drug metabolism. It is possible to detect mutations in 5 regions, and to determine, predict or test drug metabolism due to a decrease in enzyme activity.
[0011]
In the present invention, since the genetic mutation to be detected has been clarified and specified, according to the disclosure of the present invention, a method for the detection is appropriately employed or the method is appropriately modified and employed. Is easy for those skilled in the art. For example, the detection of the specific mutation (Y486D mutation) of the present invention for the subject's UGT gene can be performed according to various methods for analyzing the base sequence including the mutation position. This includes, for example, Southern hybridization method, dot hybridization method (see J. Mol. Biol., 98: 503-517, 1975, etc.), dideoxy base sequencing method, and various amplification methods combining DNA amplification methods. [For example, PCR-restriction fragment length polymorphism (RFLP), PCR-single-chain conformation polymorphism analysis (Proc. Natl. Acad. Sci., USA, 86: 2766-2770, 1989), PCR-specific sequence oligonucleotide method (SSO), allele-specific oligonucleotide method using PCR-SSO and dot hybridization method (Nature, 324: 163-166, 1986, etc.) )] And the like. Furthermore, it can be easily detected by a nucleic acid chip or a nucleic acid array using an oligonucleotide probe.
[0012]
(probe)
Mutation of 226th nucleobase in exon 1 of gene encoding UGT1 molecule (G71R), mutation of 686th nucleobase in exon 1 (P229Q) and mutation of 1456th nucleobase in exon 5 (Y486D) Can be detected using a nucleic acid probe.
The number of nucleotides of the DNA fragment for these nucleic acid probes should be at least 8, usually 10 to 50, preferably 15 to 35. If the number of nucleotides of the probe is too much more than the above, it will be difficult to hybridize to single-stranded DNA, and conversely if it is too small, the specificity of hybridization will be reduced.
[0013]
Specifically, as a G71R mutation detection probe, (SEQ ID NO: 2) an oligonucleotide having a base sequence of TCAGAGACNGAGCATTTTT, a P229Q mutation detection probe is (SEQ ID NO: 3) an oligonucleotide having a base sequence of TAATTCCCCNGTATGAA, and detection of a Y486D mutation As the probe (SEQ ID NO: 1), an oligonucleotide having a base sequence of TGGTACGCNACCATTCCT can be used. In this case, N is any of A, T, C or G or a universal base such as inosine.
Moreover, necessary chemical substances, for example, substances for facilitating spotting when preparing a nucleic acid array, various labeling substances, and the like can be added to the ends of these oligonucleotides.
[0014]
Furthermore, in the present invention, as long as it can function as a probe for UGT gene detection, it is not limited to the DNA fragment having the base sequence represented by SEQ ID NO: 1 to 3, and a small number of mismatches with the template strand. There may be. For example, as long as it has the said function, the base sequence represented by sequence number 1-3 can include the sequence | arrangement by which the modification by substitution, deletion, and / or addition of 2 or less nucleotides was carried out, for example .
[0015]
Each oligonucleotide of the probe used in the present invention can be easily synthesized according to a conventional method using an automatic synthesizer, for example, a DNA synthesizer (Perkin Elmer), etc. Further, it can be purified using a commercially available purification cartridge or the like. When the synthetic oligonucleotide is applied to a nucleic acid array immobilized on the surface of a slide glass, it is also preferable to amino-label the 5 ′ end.
[0016]
(Nucleic acid array)
Each of the above probes can be immobilized on the surface of a slide glass or the like and used as a nucleic acid array (generally also referred to as “microarray”). A publicly known method can be applied to the method of the nucleic acid array, and the method is not particularly limited (for example, under a genetic engineering experiment note, Yodosha, 175-187 (2002)). For example, it can be prepared by spotting on an aminosilane-coated slide glass using a commercially available arrayer, Affymetrix 417 Arrayer.
(Preparation of target sample and test sample)
According to the method of the present invention, drug metabolism due to glucuronidation, which occupies an important position in a kinetic test in drug development, can be easily determined. Thereby, the suitability of metabolism of the medicinal product to be administered can be determined. The sample to be measured is not particularly limited as long as it is a biological sample, and examples thereof include liver, kidney, leukocyte, organ such as hair, tissue, and the like. When the expression level of the UGT gene is small in the measurement sample, it is also possible to measure a sample obtained by amplifying the target nucleic acid by an appropriate amplification method such as PCR method, LAMP method, LCR method, NASBA method or the like. .
[0018]
A test sample can be prepared by extracting DNA from a test target sample and amplifying the DNA by, for example, PCR using a primer pair specific to the region where mutation is to be detected. Specifically, a fluorescently labeled sample is prepared using a primer (for example, 5′-Cy3 labeled oligo DNA) fluorescently labeled at the 5 ′ end, so that the prepared sample is hybridized with the probe on the nucleic acid array. The result of the hybridization can be directly detected.
[0019]
As a primer for sample preparation, for example, the following can be used. Thereby, DNA can be amplified and a fluorescence labeled sample can be prepared.
1) Primer pair for mutation detection site G71R:
G71R-F
(CTGCAGCAGAGGGGGACATGA) (SEQ ID NO: 4)
Cy3-G71R-R
(Cy3-AACATTAGCCCCGAGACTAC) (SEQ ID NO: 5)
2) Primer pair for mutation detection site P229G:
P229Q-F
(CAACCCATTCTCTCTACTGTG) (SEQ ID NO: 6)
Cy3-P229G-R
(Cy3-AGATGCAGAGCTCAATAGGTC) (SEQ ID NO: 7)
3) Primer pair for mutation detection site Y486D
a) Y486D-F
(GCTGGACCTGGCAGGTGTTC) (SEQ ID NO: 8)
Cy3-Y486D-R
(Cy3-TTTCCGGGTAGCCATTGCACA) (SEQ ID NO: 9)
b) Y486D-F2
(CCCGCAGCCCACGACCTCACCTGGT) (SEQ ID NO: 10)
Cy3-Y486D-R2
(Cy3-AGAGGAAACCAATCACGTCCAAGG) (SEQ ID NO: 11)
[0020]
(detection)
A commercially available detection apparatus is used for the detection of the nucleic acid array fluorescent label. For example, the fluorescence signal of each spot can be detected by scanning using Affymetrix 428 ArrayScanner.
[0021]
(Target drug)
The target drug that can be tested by the method of the present invention is useful for testing its metabolism as long as it is glucuronidated by UGT. Examples of these drugs include diuretics such as spironolactone, analgesics such as acetaminophen, aspirin, fructaphenine, indomethacin, psychotropic drugs such as haloperidol, carpipramine, lorazepam, amoxane, and anesthetic analgesics such as morphine, propofol, and opium , Anticancer drug doxorpidine hydrochloride, antitussive codeine phosphate, asthma drug orciprenaline sulfate, antiepileptic drug phenytoin, antihistamine drug ketotifen fumarate, antihypertensive and angina treatment drug carvedilol, propranolol hydrochloride, etc. Examples include clofibrate aluminum and Alzheimer's therapeutic drug donepezil hydrochloride.
[0022]
【Example】
EXAMPLES Hereinafter, although an Example demonstrates this invention further more concretely, this invention is not limited to these.
[0023]
[Example 1]
(Preparation of nucleic acid array)
5′-amino modified oligonucleotides having the respective sequences of SEQ ID NOS: 1 to 12 were synthesized and spotted on the surface of an aminosilane-coated slide glass (manufactured by Sigma) using an Affymetrix 417 Arrayer to prepare a DNA chip. Five of each oligonucleotide was spotted. The spot layout of the prepared array is shown in FIG.
[0024]
[Example 2]
(Sample preparation)
Seven types of test samples were PCR amplified using the following 5′-Cy3 labeled oligonucleotides and unlabeled oligonucleotide primers to prepare fluorescently labeled samples. As a primer pair for the mutation detection site G71R, a PCR amplification product of 150 base pairs using G71R-F and Cy3-G71R-R, a primer pair for the mutation detection site P229G, P229Q-F and Cy3-P229G-R are used. The PCR amplification product of 195 base pairs was further obtained using the primer pair for mutation detection site Y486D, Y486D-F and Cy3-Y486D-R. The conditions for PCR are as follows.
[0025]

The reaction product was purified by ethanol precipitation.
[0026]
[Example 3]
(Hybridization and detection)
Each sample prepared in Example 2 was mixed with 12 μL of hybridization buffer [4 × SSC (4 × represents a 4-fold concentration of 0.15 mol / L NaCl and 15 mmol / L sodium citrate as a basic solution), 0.2% SDS, 50% formamide], and 10 μL thereof was hybridized with the nucleic acid array prepared in Example 1 at 42 ° C. for 2 hours. After hybridization, wash with buffer (2 × SSC, 0.2% SDS) at 37 ° C. for 5 minutes, then wash with buffer (0.2 × SSC) at room temperature for 5 minutes, dry, and use Affimetrix 428 ArrayScanner. Was used to detect the fluorescent signal. The results are shown in FIGS.
[0027]
In sample 1, hybridization was observed only in the G row of the G71R mutation detection chip, only in the C row of the P229Q mutation detection chip, and only in the T row of the Y486D mutation detection chip. This indicated that all had a normal sequence (FIG. 2).
[0028]
In samples 2 and 3, hybridization was observed in the C row of the P229Q mutation detection chip and the T row of the Y486D mutation detection chip, and normal sequences were observed, respectively, but the A and G rows of the G71R mutation detection chip were detected. Hybridization was observed in both rows. Thereby, a heterozygous mutation having both a normal sequence hybridizing to the G column and a mutant sequence hybridizing to the A column was confirmed (FIGS. 3 and 4).
[0029]
In sample 4, the P229Q and Y486D mutation detection chips showed normal hybridization patterns, but hybridization was observed only in the A column of the G71R mutation detection chip. Thereby, a homozygous mutation was confirmed (FIG. 5).
[0030]
In sample 5, the hybridization patterns of the G71R and Y486D mutation detection chips were both normal, but in P229Q, hybridization was observed in the normal C row and further in the A row. Thereby, heterozygous mutation was confirmed (FIG. 6).
[0031]
Sample 6 showed a normal hybridization pattern for the G71R and P229Q mutation detection chips, but for the Y486D mutation detection chip, the normal T row and the G row showing mutations. Hybridization was observed in both. Thereby, heterozygous mutation was confirmed (FIG. 7).
[0032]
In sample 7, unlike sample 6, hybridization was observed only in the G row on the Y486D mutation detection chip. Thereby, a homozygous mutation was confirmed (FIG. 8).
[0033]
UGT molecules having the same gene sequences as those of the above seven samples were prepared by genetic recombination, and the UGT activity when measured using bilirubin as a substrate was compared with the above results. As a result, as shown in Table 1, UGT molecules It was confirmed that the enzyme activity of the UGT molecule can be predicted by detecting mutations in the exon 5 region that encodes, particularly Y486D mutation. This indicates that it is possible to examine abnormalities in drug metabolism. Furthermore, it was shown that abnormalities in drug metabolism can be examined more effectively by detecting mutations in exon 1 to 4 regions other than exon 5 region.
[0034]
(Table 1) UGT activity of genetically modified UGT1A1 molecule corresponding to the analysis result with DNA chip
[Table 1]

[0036]
[Example 4]
For the UGT1A6 molecule that was operated in the same manner as in Example 1 and detected a mutation in the exon 5 region, the analysis result of the mutation in the exon 5 region and the UGT activity of the corresponding recombinant UGT1A6 molecule were expressed as 2-amino-5-nitro. Measurement was carried out using -4-trifluoromethylphenol as a substrate. The results are shown in Table 2.
[0037]
(Table 2) UGT activity of genetically modified UGT1A6 molecule corresponding to the analysis result with DNA chip
[Table 2]

[0039]
As a result, it was found that not only UGT1A1 but also other isoform mutations could be detected by the method of the present invention, and the effects of the present invention were confirmed.
[0040]
【The invention's effect】
As described above, the method for detecting mutations in the common exon region encoding UGT1 of the present invention can detect a large number of mutations in the UGT1 gene efficiently without individually examining a large number of isoforms. It is possible to efficiently determine, predict and test the metabolism of a drug.
[0041]
[Brief description of the drawings]
FIG. 1 shows the arrangement of nucleic acid probes on a DNA chip.
FIG. 2 is a diagram showing a test result of sample 1 (normal specimen).
FIG. 3 is a diagram showing a test result of Sample 2. FIG.
FIG. 4 is a diagram showing a test result of a sample 3;
FIG. 5 is a diagram showing a test result of a sample 4;
FIG. 6 is a diagram showing a test result of a sample 5;
FIG. 7 is a diagram showing a test result of a sample 6;
FIG. 8 is a diagram showing a test result of a sample 7;
[Sequence Listing]

Claims (6)

A step of detecting a mutation in exon 5 region of a gene encoding UDP-glucuronosyltransferase (UGT), wherein the mutation is located at the 1456th base of the UGT gene sequence encoding the 486th amino acid of the UGT1A1 molecule. Corresponding mutations in exon 5 region of each isoform of UGT1A molecule, without detecting each mutation of UGT1A1, UGT1A3, UGT1A4, UGT1A5, UGT1A6, UGT1A7, UGT1A8, UGT1A9 and UGT1A10 A test method for drug metabolism ability of UGT. The test | inspection method of Claim 1 including the process of detecting the variation | mutation of a promoter region. The test method according to claim 1 or 2, comprising a step of detecting a mutation in at least one region of exons 1, 2, 3 and 4 of a gene sequence encoding a UGT molecule together with the step of detecting the mutation. A step of detecting a mutation in at least one gene sequence among the 226th mutation of the UGT gene sequence encoding the 71st amino acid of the UGT1A1 molecule and the 686th mutation of the gene sequence encoding the 229th amino acid. The inspection method according to claim 3, comprising: The test method according to any one of claims 1 to 4, wherein the probe having the base sequence represented by SEQ ID NO: 1 and the probe having the base sequence represented by SEQ ID NO: 2 and / or 3 are used in combination. The inspection kit used for the method of any one of Claims 1-5 .

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